Viral host range evolution has produced several emerging infectious diseases. Despite increased research on viral evolution, understanding selective pressures shaping expansion, contraction, or shift in viral host ranges remains a fundamental challenge. The proposed research will advance our objective of understanding how evolution of viral life history traits, in various multi-host environments, drives specialized or generalized use of hosts. To achieve this goal, we propose three Specific Aims: (1) developing novel mathematical models of viral host range evolution by invoking adaptive dynamics, (2) testing selection gradients predicted by the models against viral strain-competition experiments, and testing quantitative predictions of viral host range via long-term experimental evolution, and (3) exploring the role of intermediate hosts as stepping stones for infection of genetically distant hosts. For Specific Aim 1, our models will investigate evolution of viral life history traits when viral mutations feedback on selection pressures through their direct effects on the population dynamics of susceptible host densities. The theory's predictions will be tested experimentally in Specific Aim 2. We shall conduct experiments where paired viral strains compete, to determine fitness consequences of viral life history traits in various two-host environments. We further plan long-term evolution experiments to test model predictions concerning selection for specialized or generalized use of host species. To achieve our goals, we will capitalize on our extensive experience and the wealth of information available for the model system of bacterium E. coli and phage lambda. We shall construct a panel of isogenic bacterial strains differing in their recognition by phage (via changes in the receptor LamB) and differing in profitability when infected (via changes in the host NusA, NusB, and Lon protease). Furthermore, we shall construct a panel of isogenic phage strains that would differ in their ability to recognize and attach to the bacterial hosts (via chanes in the tail fiber J and side tail fiber Stf) and in their efficiency in producing phage progeny (vi changes in the antiterminator N and regulatory region nutBoxA). Lastly, in Specific Aim 3, we shall investigate the effects of host genetic distance on the likelihood of viral host jumps. Specifically, we are interested in how genetically intermediate hosts may serve as stepping stones for viral infection of genetically distant hosts. We will take advantage of the E. coli strans constructed for Specific Aim 2 by assembling an array of bacterial hosts with different levels of genetic diversity at the host factors (proteins) critical for phage infection, and by manipulating the number of host factors forming the infection barrier. Hypotheses on the means and variances of the timing of host-range mutant emergence will be tested experimentally. Our experimental design simulates spill-over infections commonly found in zoonotic viral diseases. Because of the fundamental features shared among all viruses, insights and lessons gained from this proposed research should be applicable to all other viral diseases as well. PUBLIC HEALTH RELEVANCE: Evolution of viral host range has played a major role in the emergence and epidemic spread of several important infectious diseases. We propose research seeking a new understanding of selective pressures driving the evolution of viral life history traits - traits governing host-rang evolution. Insights gained from this study should enable better prediction of viral disease emergence.